Background
Ras homolog enriched in brain (RHEB) is a member of the Ras superfamily of small GTPases that are responsible for the activation of numerous important signaling pathways in the cell [
1]. RHEB was discovered as a gene expressed in neuronal cells after synaptic stimulation and in the hippocampus after seizures [
2]. Later studies revealed RHEB to be expressed ubiquitously in all tissues [
3‐
6]. RHEB is a 21 kDa protein that is 37% identical to KRAS, and shares important features common to small GTPases including five guanine nucleotide binding domains, 6/9 identical amino acids of the Ras effector domain, and a C-terminal CAAX motif that is post-translationally farnesylated [
2,
7]. RHEB, like all small GTPases, act as molecular switches in the cell; they switch “on” and activate downstream signaling when bound GTP, and they switch “off” when bound GDP through GTP hydrolysis [
7]. However, structural studies have revealed key differences between RHEB and other members of the Ras superfamily of GTPases [
8‐
10]. A conserved amino acid important for GTP hydrolysis, Gln64 (Gln61 in Ras), is buried in a hydrophobic core blocking access to GTP [
8]. These unique structural differences cause RHEB to exist in an active GTP-bound state at higher levels than most small GTPases.
Analysis of cancer genomic databases has revealed a reoccurring point mutation in RHEB at tyrosine 35. This mutation has been identified in three patients with clear cell renal cell carcinoma (ccRCC) and two patients with endometrial cancers [
11]. RHEB Y35N was found to be significant in ccRCC due to its relatively high mutation rate relative to background and the location of the mutation in an evolutionarily conserved site [
11]. Tryosine 35 is present in the highly-conserved effector domain region of small GTPases, a region that facilitates interaction with downstream proteins and signaling activation. It is possible due to the location of this mutation, that it alters RHEB interaction with proteins and therefore alters downstream RHEB signaling pathways. Interestingly, RHEB Y35N exerts transforming effects on NIH3T3 cells as strong as that observed with KRAS G12 V transforming mutant, and this involves ERK signaling [
12].
Early studies on RHEB looked at the ability of RHEB to stimulate Ras effectors mainly due to the strong similarities between RHEB and Ras effector domains. It was demonstrated that purified RHEB could interact with RAF-1 in vitro or in a yeast two-hybrid assay [
4,
13]. Later studies indicated that RHEB binds BRAF and inhibits BRAF activation of the RAF/MEK/ERK signaling pathway [
14‐
16]. However, biological significance of the RHEB/RAF interaction was not fully explored. Concurrent studies revealed RHEB to activate mTORC1 signaling and the field of RHEB research shifted significantly to the study of mTOR [
17,
18]. mTORC1 is a kinase complex that stimulates protein synthesis and cell proliferation [
19]. Aberrant RHEB/mTORC1 signaling has been linked to many overgrowth diseases including Lymphangioleiomymoatosis (LAM), Tuberous Sclerosis (TS), Peutz-Jeghers syndrome (PJS) and benign tumor formation [
20‐
22].
We, as well as others, have continued to explore identification of downstream effectors of RHEB, as many GTPases have been shown to interact with multiple downstream effectors [
23]. In fact, the presence of multiple downstream effectors is a common feature of the RAS superfamily GTPases. For example, RAS has been shown to activate PI3K, RalGDS, RIN1, RAF, and PKC [
24]. Recent publications have linked RHEB to diverse cellular pathways through interactions with AMPK, phospholipase D1 (PLD1), β-secretase (BACE1), PDE4D, and GAPDH [
25‐
29]. Our group recently discovered a novel RHEB interaction with carbamoyl-phosphate synthetase II, aspartate transcarbamoylase, and dihydrooorotase (CAD), resulting in stimulation of pyrimidine nucleotide biosynthesis in the cell [
30]. As described in this paper, BRAF can be added as another downstream effector of RHEB.
Above developments concerning RHEB prompted us to re-evaluate RHEB-RAF interaction. In this paper we report a strong interaction between RHEB and BRAF that results in decreased BRAF-CRAF dimerization and decreased RAF/MEK/ERK signaling. This relationship is dependent on an intact effector domain and the GTP loading status of RHEB. Additionally, the Y35N mutation decreases RHEB-BRAF interaction, resulting in increased BRAF-CRAF dimerization and activation of RAF/MEK/ERK signaling. Cell lines stably expressing RHEB Y35N exhibit cancer transformation properties similar to KRAS G12 V. This evidence suggests that RHEB regulates the RAF/MEK/ERK pathway from aberrant overactivation.
Methods
Cell culture and transfection
HEK293T and NIH 3 T3 cells were obtained from ATCC (ATCC Numbers CRL-3216 and CRL-1658, respectively). HEK293T and NIH 3T3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum and 1% (vol/vol) penicillin/streptomycin. Cells were cultured at 37 °C in a 5% CO2 incubator. Transfection was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
FLAG Immunoprecipitation
HEK293T cells expressing FLAG tagged RHEB -WT, −T38A, -Y35N, -D60I, and KRAS-G12V were immunoprecipitated using anti-FLAG M2 magnetic beads (Sigma). Briefly, the cells were lysed with lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.4% CHAPS, 1X Complete EDTA-free protease inhibitor cocktail (Roche), 1 mM Na3VO4), 150 mM NaCl, 25 mM MgCl2), and the supernatant was cleared of cellular debris using centrifugation (16,000×g for 10 min). Cleared supernatant was mixed with anti-FLAG M2 magnetic beads (Sigma) for affinity purification. The beads were collected, washed four times with lysis buffer. The remaining bound proteins were eluted three times with lysis buffer containing 62 μg/mL of 3X FLAG peptide. Eluted proteins were concentrated using Amicon Ultra 0.5-ml centrifugal filters NMWL 10 K (EMD Millipore, Billerica, MA).
Western blotting & antibodies
The amount of total protein concentration in cellular lysate was determined by Bio-Rad protein assay according to manufacturer’s instructions. Western blotting was carried out as described previously [
31]. Briefly, equal protein extracts from samples were separated by SDS-PAGE and transferred onto nitrocellulose membrane (GE Healthcare). The membrane was blocked in 5% bovine serum albumin, incubated in primary antibodies, and followed by incubation in secondary antibodies conjugated to Horseradish peroxidase (HRP). The membrane was incubated in Pierce ECL Western Blotting Substrate solution (Thermo Scientific) to activate the HRP activity, and protein bands were detected on film.
The following antibodies were purchased from Cell Signaling Technologies: Anti –RHEB, -KRAS, -ACTIN, -totalS6, -phosphoS6, -totalERK, -phosphoERK, -BRAF, and –CRAF. Anti-FLAG was purchased from Sigma.
Generation of Lentivirus and stably expressing cell lines
Stably expressing cell lines were generated using lentiviral transduction method. The RHEB and KRAS G12V genes were amplified from pcDNA.3 plasmid vectors already containing Flag-RHEB or Flag-KRAS G12V via PCR, and primers containing EcoRI and BamHI restriction enzyme cut sites. Amplified products were ligated into the lentiviral transfer plasmid pCCL-c-MCS, after it was digested with EcoRI and BamHI, using ligase. The RHEB Y35N mutation was generated using Quickchange Lighting Site-Directed Mutagenesis Kit (Agilent).
Lentivirus was produced by transfecting the lentiviral transfer plasmid, the packaging plasmid (pCMV-R8.9) and the envelope plasmid (pMDG-VSVG) into HEK 293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The media was collected 48 h later and filtered through a 0.45 μm filter. Lentiviral media was stored at −80 °C until ready for use.
NIH 3T3 cells were grown until 90% confluency before adding a mixture of 50% normal media, 50% lentiviral media, and 8 μg/mL polybrene. Cells were incubated for 48 h before being passaged and grown in normal media. Expression of transduced proteins were monitored via Western blot using anti-FLAG antibodies.
Growth curve assay
Cells were grown under normal conditions (DMEM containing 10% FBS) or serum starved (DMEM without FBS) and measured at given timepoints using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc.) according to manufacturer’s instructions. Briefly, cells were grown in 96 well plates, 10 μl of CCK-8 dye was added to each well containing 100 μl of cell media, cells were incubated for 1 h, and then readings were obtained in triplicates using a Spectramax Plus 384 spectrophotometer (Molecular Devices) at O.D. 450 nm.
Cell cycle analysis
NIH 3T3 cell lines were trypsinized, washed, and suspended in PBS. Cells were fixed for 1 h at 4 °C in 70% ethanol. After fixation, cells were washed of ethanol and suspended in 500 μl of PBS. 20 μl of RNAase A (10 mg/mL stock) and 25 μl of propidium iodide (1 mg/mL stock) solutions were added and the cells were incubated at 37 °C for 30 min. Cells were analyzed by flow cytometry at the UCLA Flow Cytometry Core.
NIH 3T3 cell lines were grown under normal growth conditions for 3 weeks, fresh media was added every 2-3 days. Cells were visualized with crystal violet staining method. Briefly, cells were fixed with ice-cold methanol for 10 min on ice. Methanol was removed and the cells were incubated in 0.5% crystal violet solution (0.5 g crystal violet in 100 ml of 25% methanol solution) for 5 min at room temperature. Cells were rinsed with H2O until no more color came off in the rinse. For quantification, only those foci that were > than 2.5 mm in diameter were counted.
To generate a semi-solid media growth surface for cells, first a 1% and a 0.6% (mass/vol) agar-media solution was made and autoclaved. Then a 0.5% base-layer-matrix was generated by heating up the 1% agar solution until dissolved, and mixing it with normal growth media in a 50:50 ratio. The solution was layered onto a cell culture plate and left to solidify in the cell incubator for 1 h. The 0.6% agar solution was heated until dissolved, and placed in 37 °C H2O bath to bring down to cell temperature. The NIH 3T3 cell lines were trypsinized and suspended in normal media and the 0.6% agar solution in a 50:50 ratio (now a 0.3% agar-media-cell solution). The 0.3% agar-media-cell solution was layered on top of the 0.5% solidified base-layer-matrix. Cells were grown in incubator as normal for 3–4 weeks, with small amount of normal media added 1×/week to prevent the gels from drying out. Cells were incubated with Nitro Blue Tetrazolium dye 1 mg/ml stock (tablets purchased from Sigma) overnight at 37 °C. Colonies were visualized using BioRad Imager and counted by eye.
Discussion
In this paper, we have shown that RHEB interacts with BRAF. Use of two RHEB mutants, T38A and D60I established that this interaction is dependent on the intact effector domain as well as on GTP binding status. Thus, BRAF is a downstream effector of RHEB. On the other hand, we have not detected interaction of RHEB with CRAF, suggesting that RHEB specifically interacts with BRAF. The reason RHEB binds BRAF and not CRAF needs further investigation. BRAF and CRAF are very similar in homology, with only a few differences between them. Most notably, BRAF has an extended portion of the N-terminus that is not present in CRAF. It has been reported that this extra N-terminal sequence facilitates RAS binding with BRAF differently than with CRAF [
40]. It could be that this is the area where RHEB interacts, but further studies are needed to determine the RHEB binding site on BRAF. We further showed that RHEB inhibits BRAF-CRAF dimer formation.
Significance of RHEB-BRAF interaction was further supported by the experiment to knockdown RHEB. Increased ERK signaling was observed when RHEB expression was inhibited by shRNA. In contrast, overexpression of RHEB results in the inhibition of the ERK signaling. Thus, RHEB suppresses the ERK signaling through its interaction with BRAF and inhibition of the formation of BRAF-CRAF heterodimer.
The oncogenic RHEB mutant Y35N was identified in human cancers including renal cancer and endometrial cancer [
11]. We have shown the oncogenic RHEB mutant, RHEB Y35N, interacts less efficiently with BRAF when compared with the wild type RHEB. Furthermore, the Y35N mutant does not inhibit BRAF-CRAF heterodimerization, while the wild type RHEB does. Thus, ERK signaling is sustained at a higher level in mutant cells than in wildtype, contributing to transformation. On the other hand, the RHEB Y35N mutant behaves similarly to the wild type with respect to the activation of mTORC1 signaling. We also examined the binding of the mutant RHEB Y35N to AMPK, as a previous report suggested that RHEB Y35N transforms cancer cells through an interaction with AMPK [
12]. This paper argues that RHEB Y35N displays stronger binding to AMPK, which prevents AMPK from phosphorylating and inhibiting BRAF. However, in our experiments we did not observe increased binding of RHEB Y35N to AMPK when compared with the RHEB WT (Additional file
3: Figure S1).
Transforming capability of the RHEB Y35N mutant was evaluated by establishing a stable cell line expressing the mutant RHEB. We find that these cells exhibit serum independent growth; they avoid G1 cell cycle arrest and continue to grow in the absence of serum. These cells also exhibit foci formation and soft agar growth demonstrating anchorage independent growth. Strikingly, the transforming ability of the RHEB mutant was as strong as that of the KRAS G12V mutant. In further support of the significance of the increased ERK signaling and not mTORC1 signaling in the Y35N expressing cells, proliferation of these cells were inhibited by MEK inhibitor but not by rapamycin.
Presence of multiple downstream effectors is a common feature of the Ras superfamily GTPases, as evidenced by identification of multiple downstream effectors of RAS that includes RAF, PI3K, RalGDS, RIN1 and PKC. Our current study firmly establishes that BRAF is a critical downstream effector of RHEB. Since it has been established that mTORC1 is a downstream effector of RHEB, RHEB affects multiple downstream signaling pathways. Further work on RHEB signaling could define the significance of these downstream signaling pathways and in turn define the function of RHEB GTPase.
Acknowledgements
Flow cytometry was performed in the UCLA Jonsson Comprehensive Cancer Center (JCCC) and Center for AIDS Research Flow Cytometry Core Facility that is supported by National Institutes of Health awards P30 CA016042 and 5P30 AI028697, and by the JCCC, the UCLA AIDS Institute, and the David Geffen School of Medicine at UCLA.